Genes for detecting bacteria and detection method by using the same

ABSTRACT

The present invention relates to genes for detecting the genus  Pectinatus frisingensis  or  Pectinatus cerevisiiphilus  of the genus Pectinatus, which is known as beer-spoilage bacteria, and a method for detecting the bacteria by using the genes. 
     The present invention provides gene sequences of spacer regions between 16S rRNA genes and 23S rRNA genes specific for the genus Pectinatus relating to beer-spoilage and a method for quickly and sensitively detecting the bacteria by using the sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is filed under 35 U.S.C. 317(c) (1) as the U.S.National phase of PCT Application PCT/JP99/04341, filed Aug. 11, 1999,which claims priority from Japanese application 10/227177, filed Aug.11, 1998.

FIELD OF THE INVENTION

The present invention relates to genes for detecting Pectinatusfrisingensis or Pectinatus cerevisiiphilus of the genus Pectinatus,which is known as beer-spoilage bacteria, and a method for detecting thebacteria by using the genes.

DESCRIPTION OF THE PRIOR ART

Bacteria of the genus Pectinatus have been known as beer-spoilagebacteria. In the genus, two kinds of Pectinatus frisingensis andPectinatus cerevisiiphilus have been known. For detecting the bacteriaof the genus Pectinatus, the bacteria must be isolated aftermultiplication culture and separation culture. It takes at least sevendays. Then, isolated bacteria are multiplied and tested by manyqualitative tests such as morphological observation, gram stainability,a catalase test, utilization of various carbon sources and the like toidentify the bacteria.

These tests are very troublesome, and it takes much time and it costsmuch. In addition to these common identification tests, there is amethod that DNA is extracted from isolated bacteria, fixed on amembrane, and conducted a hybridization test by using standard bacteriaDNA as a probe to identify the class. However, it takes some days, andit is difficult to obtain necessary detective sensitivity andselectivity.

Lately, a method for detection of bacteria of the genus Pectinatus isdisclosed by using a monoclonal antibody that specifically reacts withPectinatus cerevisiiphilus (ASBC Journal: 51(4)158-163, 1993). However,the method is insufficient to the detective sensitivity. The method hasa problem that Pectinatus frisingensis can not be detected.

The other detection method has been reported. It can detect Pectinatusfrisingensis and Pectinatus cerevisiiphilus by a Ribotyping method thatpolymorphism of a ribosomal RNA gene is detected (J. Am. Soc. Chem.: 56(1) 19-23, 1998). However, since the method needs operation forisolating the bacteria, it has problems of detective sensitivity andspeed.

Considering these problems, further quick detection methods have beenstudied. W097/20071 discloses a method for detecting Pectinatuscomprising extracting DNA of the test microorganism, and using a PCRmethod that a complementary oligonucleotide of the DNA functionates as aprimer. However, the base sequences of 16S rRNA gene used in thetechnique are sometimes similar to those of microorganisms of the othergenera, so that there are problems that the other microorganisms aredetected in addition to particular microorganisms to be detected.

The gene in a spacer between a 16S rRNA gene and a 23S rRNA gene has aspecific gene sequence. Though methods for detecting microorganismsusing the gene sequence are disclosed in Japanese Jozo Ronbunshu 50,22-31 (1995), APPL. ENVIRON. MICROBIOL. VOL.62, NO.5, 1683-1688(1996),FEMS MICROBIOL LETT. VOL. 84, NO.3, 307-312(1991), Japanese Patent KokaiPublication No. 6-98800 and the like, gene sequences of the spacers ofthe genus Pectinatus have not been found.

SUMMARY OF THE INVENTION

The present invention aims to provide gene sequences of a spacer regionthat is constituted between a 16S rRNA gene and a 23S rRNA gene specificfor the genus Pectinatus relating to beer-spoilage, and to provide amethod for sensitively and quickly detecting the genus by using thesequences.

(1) The first invention is a gene sequence of a spacer region between agene coding 16S rRNA and a gene coding 23S rRNA of Pectinatusfrisingensis containing a part of the base sequence or the whole basesequence represented by SEQ ID NO: 1.

(2) The second invention is a gene sequence of a spacer region between agene coding 16S rRNA and a gene coding 238 rRNA of Pectinatusfrisingensis containing a part of the base sequence or the whole basesequence represented by SEQ ID NO: 2.

(3) The third invention is a gene sequence of a spacer region between agene coding 16S rRNA and a gene coding 23S rRNA of Pectinatuscerevisiiphilus containing a part of the base sequence or the whole basesequence represented by SEQ ID NO: 3.

(4) The fourth invention is a gene sequence of a spacer region between agene coding 16S rRNA and a gene coding 23S rRNA of Pectinatuscerevisiiphilus containing a part of the base sequence or the whole basesequence represented by SEQ ID NO: 4.

(5) The fifth invention is an oligonucleotide characterized in that thegene sequence of a spacer region between a gene coding 16S rRNA and agene coding 23S rRNA of Pectinatus frisingensis has at least one of thefollowing sequence group or the corresponding complementary sequence:

5′-CCATCCTCTTGAAAATCTC-3′{circle around (1)} (SEQ ID NO:5)

5′-TCTCRTCTCACAAGTTTGGC-3′{circle around (2)} (SEQ ID NO:6)

(6) The sixth invention is an oligonucleotide characterized in that thegene sequence of a spacer region between a gene coding 16S rRNA and agene coding 23S rRNA of Pectinatus cerevisiiphilus has at least one ofthe following sequence group or the corresponding complementarysequence:

5′-CACTCTTACAAGTATCTAC-3′{circle around (3)} (SEQ ID NO:7)

5′-CCACAATATTTCCGACCAGC-3′{circle around (4)} (SEQ ID NO:8)

5′-AGTCTTCTCTACTGCCATGC-3′{circle around (5)} (SEQ ID NO:9)

(7) The seventh invention is a method for detecting Pectinatusfrisingensis, wherein the oligonucleotide made from the gene sequencedescribed in (1) or (2) uses as a primer for synthesis of nucleic acids,and the nucleic acid is treated by gene amplification to detect thebacteria.

(8) The eighth invention is a method for detecting Pectinatuscerevisiiphilus, wherein the oligonucleotide made from the gene sequencedescribed in (3) or (4) uses as a primer for synthesis of nucleic acids,and the nucleic acid is treated by gene amplification to detect thebacteria.

(9) The ninth invention is a method for detecting Pectinatusfrisingensis, wherein the oligonucleotide made from the gene sequencedescribed in (1) or (2), or the oligonucleotide made from the genesequence described in (5), and a nucleotide sequence coding 16S rRNAgene of Pectinatus frisingensis use as primers for synthesis of nucleicacids, and the nucleic acid is treated by gene amplification to detectthe bacteria.

(10) The tenth invention is a method for detecting Pectinatuscerevisiiphilus, wherein the oligonucleotide made from the gene sequencedescribed in (3) or (4) or the oligonucleotide made from the genesequence described in (6), and a nucleotide sequence coding 1 6S rRNAgene of Pectinatus cerevisiiphilus use as primers for synthesis ofnucleic acids, and the nucleic acid is treated by gene amplification todetect the bacteria.

(11) The eleventh invention is a method as in (9), wherein thenucleotide sequence coding the 16S rRNA gene of Pectinatus frisingensishas the following sequence:

5′-CGTATCCAGAGATGGATATT-3′{circle around (6)} (SEQ ID NO:10)

(12) The twelfth invention is a method as in (10), wherein thenucleotide sequence coding the 16S rRNA gene of Pectinatuscerevisiiphilus has the following sequence:

5′-CGTATGCAGAGATGCATATT-3′{circle around (7)} (SEQ ID NO:11)

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. It shows Electrophoretogram in Example 3.

FIG. 2. It shows Electrophoretogram in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

Since the technique of gene amplification is well known, it is conductedunder the polymerase chain reaction method which has been developed bySaiki et al. (abbreviated as PCR method hereinafter; Science 230, 1350,1985).

This method is conducted by amplification reaction of particular genesequences. Since the method shows quick reaction, high sensitivity andspecificity and convenience, applications has been tried to quicklyjudge viruses in medical fields or quickly detect noxious bacteria infood fields. By the PCR method, even if only a few nucleotide sequencesare present in test samples, the target nucleotide sequence between twoprimers is amplified several hundred times, and the copies are producedin large quantities to be detectable. For conducting the PCR method, thenucleic acid ingredient should be liberated from the bacteria in thetest samples. However, in the PCR method, when several or more moleculesexist in the target sequence, the amplification reaction proceeds.Accordingly, samples of the PCR method can be provided by a simplepretreatment of the bacteria with a lytic enzyme or a surfactant. Forthis reason, the method for detecting bacteria has merits higher thanconventional methods.

The present invention provides gene sequences of a spacer region betweena gene coding 16SrRNA and a gene coding 23SrRNA in each Pectinatusfrisingensis or Pectinatus cerevisiiphilus. By using a nucleotidesequence coding a 16SrRNA gene or oligonucleotide which is selected fromthe sequence as a primer for nucleic acid synthesis in the PCR method,and by gene amplification treatment, the present inventors havedeveloped a quick and high sensitive method for judging the existence ofPectinatus frisingensis or Pectinatus cerevisiiphilus in samples.

The test samples may be beer or semi-products of beer, or a sampleextracted from sewage and the like. The oligonucleotide for a primer maybe a chemical synthetic or natural product.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As shown hereinafter, in the method of the present invention, Pectinatusfrisingensis or Pectinatus cerevisiiphilus is detected by the PCRmethod. The base sequences used in the PCR method are, not by way oflimitation, for example, above-mentioned (5), (6), (11) and (12). Theprimer length used in the PCR method is, not by way of limitation,1.9-20 base length in above-mentioned (5), (6), (11) and (12),preferably, 10-50 base length.

When Pectinatus frisingensis is detected by the PCR method, theexistence of the bacteria is judged by that, the DNA fragments amplifiedin case of the combination of {circle around (1)} and {circle around(6)} as the primer are about 700 base pairs and about 900 base pairs,and the DNA fragments amplified in case of the combination of {circlearound (2)} and {circle around (6)} as the primer are about 700 basepairs and about 900 base pairs. When these bands are detected byelectrophoresis, it is judged that Pectinatus frisingensis is present.Since the combination of the primers, in any cases, is specific forPectinatus frisingensis bacteria, the genus can be detected. By parallelusing two of the combination, further precise determination becomespossible. By changing the base sequences of the primers used in the PCRmethod, the length of the nucleotide sequences amplified can be changed.

On the other hand, when Pectinatus cerevisiiphilus is detected by thePCR method, the existence of the bacteria is judged by that the DNAfragments amplified in case of the combination of {circle around (3)}and {circle around (7)} are about 600 base pairs, the DNA fragmentsamplified in case of the combination of {circle around (4)} and {circlearound (7)} are about 650 base pairs, and the DNA fragments amplified incase of the combination of {circle around (5)} and {circle around (7)}are about 700 base pairs. When these bands are detected byelectrophoresis, it is judged that Pectinatus cerevisiiphilus ispresent. Since the combination of the primers, in any cases, is specificfor Pectinatus cerevisiiphilus bacteria, the genus can be detected. Byparallel using two or more of the combination, further precisedetermination becomes possible. By changing the base sequences of theprimers used in the PCR method, the length of the nucleotide sequencesamplified can be changed.

The temperature conditions of one cycle in the PCR method are 90-98° C.in a thermal denaturation reaction in which double-stranded DNA ischanged to single-stranded DNA, 37-65° C. in an annealing reaction inwhich DNA is hybridized into primer template DNA, and 50-75° C. in achain elongation reaction in which DNA polymerase is reacted. The targetsequences can be amplified by several ten cycles. After PCR reaction,the reactant is separated by electrophoresis, and the nucleic acid isstained with ethidium bromide or the like. When the base length of theamplified nucleotide sequence is equal to the base length of the abovetarget sequence, it can be judged that the bacteria to be detected arein the test sample. To detect the amplified nucleotide sequence,chromatography is usable.

The sequences of the present invention are described in the following:

SEQ ID NO: 1 The sequence length is 624, the sequence type is nucleicacid, the strandness is double, the topology is linear, the moleculetype is genomic DNA, strandness is double, the topology is linear, themolecule type is genomic DNA, and the original source is Pectinatusfrisingensis DSM6306.

SEQ ID NO: 3 The sequence length is 724, the sequence type is nucleicacid, the strandness is double, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatuscerevisiiphilus DSM20467.

SEQ ID NO: 4 The sequence length is 399, the sequence type is nucleicacid, the strandness is double, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatuscerevisiiphilus DSM20467.

SEQ ID NO: 5 The sequence length is 19, the sequence type is nucleicacid, the strandness is single, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatus frisingensisDSM6306.

SEQ ID NO: 6 The sequence length is 20, the sequence type is nucleicacid, the strandness is single, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatus frisingensisDSM6306.

SEQ ID NO: 7 The sequence length is 19, the sequence type is nucleicacid, the strandness is single, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatuscerevisiiphilus DSM20467.

SEQ ID NO: 8 The sequence length is 20, the sequence type is nucleicacid, the strandness is single, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatuscerevisiiphilus DSM20467.

SEQ ID NO: 9 The sequence length is 20, the sequence type is nucleicacid, the strandness is single, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatuscerevisiiphilus DSM20467.

SEQ ID NO: 10 The sequence length is 20, the sequence type is nucleicacid, the strandness is single, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatus frisingensisDSM6306.

SEQ ID NO: 11 The sequence length is 20, the sequence type is nucleicacid, the strandness is single, the topology is linear, the moleculetype is genomic DNA, and the original source is Pectinatuscerevisiiphilus DSM20467.

The present invention is described by working examples in the following.The present invention is not limited by these examples.

EXAMPLE 1 Preparation of test samples

Pectinatus frisingensis DSM6306 and Pectinatus cerevisiiphilus DSM20467were used as bacterial strains belonging to Pectinatus. To confirm thespecificity of Pectinatus frisingensis and Pectinatus cerevisiiphilusprimers shown in SEQ ID NO:5, 6, 7, 8, 9, 10 and 11 in the presentinvention, the other bacteria shown in Table 1 were used. These bacteriawere cultivated on suitable culture mediums, and the strains werecollected by centrifugation. The DNA from the strains were extracted inaccordance with the description of SHIN-SEIKAGAKU-JIKKEN-KOZA 2, Nucleicacid I, Separation and Purification, p.p. 20-21 (edited by JapanBiochemical Learned Society, Tokyo-Kagaku-Dojin), and a DNA solution wasobtained.

TABLE 1 Bacteria Strain No. Bacteria type name Remarks 1 Pectinatusfrisingensis DSM6306 type strain 2 Pectinatus cerevisiiphilus DSM20467type strain 3 Selenomonas lacticifex DSM20757 type strain 4 Zymophilusraffinosivorans DSM20765 type strain 5 Zymophilus paucivorans DSM20756type strain 6 Escherichia coli IF03301 K-12 7 Megasphaera cerevisiaeDSM20462 type strain 8 Lactobacillus acidophilus IF013951 type strain 9Lactobacillus plantarum JCM1149 type strain 10 Lactobacillus brevisJCM1059 type strain 11 Lactococcus lactis JCM5805 type strain 12Leuconostoc mesenteroides JCM6124 type stpain 13 Pediococcus damnosusJCM5886 type strain

EXAMPLE 2 Cloning of spacer regions between the gene coding 16S rRNA andthe gene coding 23S rRNA of Pectinatus frisingensis, and determinationof the base sequences

(1) Selection and synthesis of oligonucleotide primers for amplificationof 16S/23S rRNA spacer region by the PCR method

Since the base sequences of the 16S ribosomal RNA gene of Pectinatusfrisingensis were apparent (International Journal of SystematicBacteriology, Vol. 40, p.p. 19-27 (1990)), the primers were selected onthe basis of the 557-576^(th) base sequences.

Since the base sequences of the 23S ribosomal RNA gene of Pectinatusfrisingensis were apparent (Systematic Applied Microbiology, Vol. 15,p.p. 487-501 (1990), EMBL Accession Number X48423), the primers wereselected on the basis of the 1-20^(th) base sequences to obtaincorresponding comprehensive sequences. The synthesis was entrusted toSawady Technology Co., Ltd.

(2) Amplification of 16S/23S rRNA spacer regions by the PCR method

The Pectinatus frisingensis DNA solution 0.1 μg, which was prepared inExample 1, was placed in a 0.2 ml tube (manufactured by Perkin-Elmer), 5μl of 10× buffer in a rTaq DNA Polymerase Kit (Toyobo Co., Ltd.), 3 μlof 25 mM MgCl₂, 5μl of a 2 mM dNTP mixture solution (dATP, dGTP, dCTPand dTTP), 0.5 μl of 5 units 1 μl of rTaq polymerase, and each 0.5 μl of100 mM primers prepared in Example 2-(1) were added to the solution, andthen sterilized distilled water was added to obtain final volume of 50μl. The tube was set on a thermal cycler of an automatic geneamplification device (Perkin Elmer) and the amplification method wasconducted. The reaction was repeated by 30 cycles, and one cycle had thefollowing conditions:

Denaturation at 94° C. for 2.5 minutes; Denaturation at 94° C. for 30seconds; Annealing of primers at 55° C. for 30 seconds; and syntheticreaction at 72° C. for 30 seconds. After the reaction, using 5 μl of thesolution, electrophoresis was conducted by agarose gel. DNA was dyedwith ethidium bromide, and amplified DNA was observed. The result showsthat, about 1600 bp (abbreviated as “long” hereinafter) DNA and about1400 bp (abbreviated as “short” hereinafter) DNA were amplified.

(3) Cloning and sequencing of the spacer region “long” Using a high purePCR product purification kit (Baringer Manhaim), unreactive dNTPs wasremoved from the solution after the PCR reaction. To the resultingamplified DNA 100 ng, 2 μl of plasmid pCR 2.1 contained in a TA cloningkit (INVITROGEN), 1 μl of ligase and 1 μl of buffer were added, and thensterilized water was added to obtain the total volume of 10 μl. Afterthe solution was reacted at 14° C. for 4 hours, 2 μl of the solution and2 μl of 0.5 M β-mercaptoethanol were added to Escherichia coli INV α′Fcompetent cells, and placed in ice for 30 minutes. Then, the solutionwas heated at 42° C. for 30 seconds, and plasmid transformation to thebacteria was conducted. To the transformed bacteria, 250 μl of a SOCculture (2.0% Tryptone, 0.5% yeast extract, 10.0 mM NaCl, 2.5 mM KCl,10.0 mM MgCl₂—6F′H₂O, and 20.0 mM glucose) was added, and the mixturewas shaken at 37° C. for 60 minutes, then transferred to a LB plateculture medium containing 50 μg/ml of ampicillin and 40 μg/ml X-Gal, andcultured at 37° C. overnight. The expressed white colony was transferredto 3 ml of a LB liquid culture medium containing 50 μg/ml of ampicillin,and cultured at 37° C. overnight.

After the cultivation, plasmids were extracted from the bacteria with aplasmid mini kit (QIAGEN). Apart of the resulting plasmids was taken outand reacted with a restriction enzyme EcoRI (manufactured by TakaraShuzo) at 37° C. for 60 minutes, and separated by agaroseelectrophoresis. The DNA was dyed with ethidium bromide, and insertionof “long” was confirmed. 500 ng of the residual plasmid was reacted withrestriction enzyme SmaI (manufactured by TOYOBO Co., Ltd.) at 30° C. for60 minutes. To the reactant, 2 μl of 3 M sodium acetate and 500 μl of100% ethanol were added, and the mixture was placed in ice for 15minutes and centrifuged at 15000 rpm for 15 minutes, and the supernatantwas removed. To the precipitate, 500 μl of 70% ethanol was added, themixture was centrifuged at 15000 rpm for 15 minutes, and the supernatantwas removed, and dried for 10 minutes under reduced pressure. Sterilizedwater was added to dissolve the precipitate, and the mixture was reactedwith restriction enzyme XbaI (Baringer Manhaim) at 37° C. for 60minutes. To the reactant, equivalent phenol/chloroform (equivalentmixture liquid) was added and gently mixed, the mixture was centrifugedat 15000 rpm for 15 minutes, and the water layer (upper layer) wasrecovered.

To the recovery liquid, equivalent water-saturated ether was added andgently mixed, and the mixture was centrifuged at 15000 rpm for 15minutes to remove the ether layer (upper layer). To the remaining waterlayer, 2 μl of 3M sodium acetate and 500 μl of 100% ethanol were added,and the mixture was placed in ice for 15 minutes and centrifuged at15000 rpm for 15 minutes to remove the supernatant. To the precipitate,500 μl of 70% ethanol was added, and the mixture was centrifuged at15000 rpm for 15 minutes to remove the supernatant, and the residue wasdried under reduced pressure for 10 minutes, and 20 μl of sterilizeddistillation water was added. To 5 μl of the solution, 1 μl of 10×buffer contained in a blunting kit (Takara Shuzo Co., Ltd.) and 3 μl ofsterilized distillation water were added, and the mixture was maintainedat 70° C. for 5 minutes, 1 μl of T4 DNA polymerase was added, and themixture was maintained at 37° C. for 5 minutes to obtain blunt ends.After T4 DNA polymerase was inactivated by stirring, 40 μl of ligationsolution A and 10 μl of ligation solution B were added, and the mixturewas maintained at 16° C. for 30 minutes to conduct internal ligation.

The reactant 2 μl and 2 μl of 0.5M β-mercaptoethanol were added to aEscherichia coli INVα′F competent cell, and the mixture was placed inice for 30 minutes and heated at 42° C. for 30 seconds, and the plasmidwas transformed to the Escherichia coli. To the transformed Escherichiacoli, a SOC culture medium (2.0% Tryptone, 0.5% Yeast extract, 10.0 mMNaCl, 2.5 mM KCl, 10.0 mM MgCl₂—6H₂O, 20.0 mM glucose) 250 μl was added,and the mixture was shaken at 37° C. for 60 minutes and spread on a LBplate culture medium containing 50 μg/ml ampicillin to culture at 37° C.overnight. Appeared white colonies were inoculated into 3 ml of a LBliquid culture medium containing 50 μg/ml of ampicillin and cultured at37° C. overnight. After the culture, the plasmid was extracted from theEscherichia coli with a plasmid mini kit (QIAGEN Company).

Using such obtained plasmid as a template, a sequence reaction wasconducted. As the sequencing primers, an IRD41 Infrared Dye Labeled M13Forward primer and an IRD41 Infrared Dye Labeled M13 Reverse primer(manufactured by Nisshinbo, sold by Aroka Co., Ltd.) were used. As thereaction liquid, SequiTherm (trademark) Long-Read (trademark) CycleSequencing Kit-LC (manufactured by EPICENTRE TECHNOLOGIES) was used.4000L Long ReadIR (trademark) DNA Sequencing System (manufactured byLI-COR) was used for the determination of the base sequences.

The gene sequence of spacer region “long” between the gene coding 16SrRNA and the gene coding 23S rRNA of Pectinatus frisingensis DSM6306bacteria is shown in SEQ ID NO: 1.

(4) Cloning and sequencing of spacer region “short”

Using a high pure PCR product purification kit (Baringer Manhaim),unreactive dNTPs was removed from the solution after the PCR reaction inExample 2-(2). To the resulting amplified DNA 1.00 ng, 2 μl of plasmidpCR 2.1 contained in a TA cloning kit (INVITROGEN), 1 μl of ligase and 1μl of buffer were added, and then sterilized water was added to obtainthe total volume of 10 μl. After the solution was reacted at 14° C. for4 hours, 2 μl of the solution and 2 μl of 0.5 M β-mercaptoethanol wereadded to Escherichia coli INVα′F competent cells, and placed in ice for30 minutes. Then, the solution was heated at 42° C. for 30 seconds, andplasmid transformation to the bacteria was conducted. To the transformedbacteria, 250 μl of a SOC culture (2.0% Tryptone, 0.5% yeast extract,10.0 mM NaCl, 2.5 mM KCl, 10.0 mM MgCl₂—6H₂O and 20.0 mM glucose) wasadded, and the mixture was shaked at 37° C. for 60 minutes, thentransferred to a LB plate culture medium containing 50 μg/ml ofampicillin and 40 μg/ml X-Gal, and cultured at 37° C. overnight. Theappeared white colony was transferred to 3 ml of a LB liquid culturemedium containing 50 μg/ml of ampicillin, and cultured at 37° C.overnight. After the cultivation, plasmid was extracted from thebacteria with a plasmid mini kit (QIAGEN).

A part of the resulting plasmid was taken out and reacted with arestriction enzyme EcoRI (manufactured by Takara Shuzo) at 37° C. for 60minutes, and separated by agarose electrophoresis. The DNA was dyed withethidium bromide, and insertion of “short” was confirmed. 500 ng of theresidual plasmid was reacted with restriction enzyme SmaI (manufacturedby TOYOBO Co., Ltd.) at 30° C. for 60 minutes. To the reactant, 2 μl of3 M sodium acetate and 500 μl of 100% ethanol were added, and themixture was placed in ice for 15 minutes and centrifuged at 15000 rpmfor 15 minutes, and the supernatant was removed. To the precipitate, 500μl of 70% ethanol was added, the mixture was centrifuged at 15000 rpmfor 15 minutes, and the supernatant was removed, and dried for 10minutes under reduced pressure. Sterilized water was added to dissolvethe precipitate, and the mixture was reacted with restriction enzymeXbaI (Baringer Manhaim) at 37° C. for 60 minutes. To the reactant,equivalent phenol/chloroform (equivalent mixture liquid) was added andgently mixed, the mixture was centrifuged at 15000 rpm for 15 minutes,and the water layer (upper layer) was recovered. To the recovery liquid,equivalent water-saturated ether was added and gently mixed, and themixture was centrifuged at 15000 rpm for 15 minutes to remove the etherlayer (upper layer).

To the remaining water layer, 2 μl of 3M sodium acetate and 500 μl of100% ethanol were added, and the mixture was placed in ice for 15minutes and centrifuged at 15000 rpm for 15 minutes to remove thesupernatant. To the precipitate, 500 μl of 70% ethanol was added, andthe mixture was centrifuged at 15000 rpm for 15 minutes to remove thesupernatant, and the residue was dried under reduced pressure for 10minutes, and 20 μl of sterilized distilled water was added. To 5 μl ofthe solution, 1 μl of 10× buffer contained in a blunting kit (TakaraShuzo Co., Ltd.) and 3 μl of sterilized distilled water were added, andthe mixture was maintained at 70° C. for 5 minutes, 1 μl of T4 DNApolymerase was added, and the mixture was maintained at 37° C. for 5minutes to obtain blunt ends. After T4 DNA polymerase was inactivated bystirring, 40 μl of ligation solution A and 10 μl of ligation solution Bwere added, and the mixture was maintained at 16° C. for 30 minutes toconduct internal ligation. 2 μl of the reactant and 2 μl of 0.5Mβ-mercaptoethanol were added to a Escherichia coli INVα′F competentcell, and the mixture was placed in ice for 30 minutes and heated at 42°C. for 30 seconds, and the plasmid was transformed to the Escherichiacoli.

To the transformed Escherichia coli, 250 μl of SOC culture medium (2.0%Tryptone, 0.5% Yeast extract, 10.0 mM NaCl, 2.5 mM KCl, 10.0 mMMgCl₂—6H₂O, 20.0 mM glucose) was added, and the mixture was shaken at37° C. for 60 minutes and spread on a LB plate culture medium containing50 μg/ml ampicillin to culture at 37° C. overnight. Appeared whitecolonies were inoculated into 3 ml of a LB liquid culture mediumcontaining 50 μg/ml of ampicillin and cultured at 37° C. overnight.After the culture, the Plasmid was extracted from the Escherichia coliwith a plasmid mini kit (QIAGEN Company).

Using such obtained plasmid as a template, a sequence reaction wasconducted. As the sequencing primers, an IRD41 Infrared Dye Labeled M13Forward primer and an IRD41 Infrared Dye Labeled M13 Reverse primer(manufactured by Nisshinbo, sold by Arok co., Ltd.) were used. As thereaction quid, SequiTherma (trademark) Long-Read (trademark) CycleSequencing Kit-LC (manufactured by EPICENTRE TECHNOLOGIES) was used.4000L Long ReadIR (trademark) DNA Sequencing System (manufactured byLI-COR) was used for the determination of the base sequences.

The gene sequence of spacer region “short” between the gene coding 16SrRNA and the gene coding 23S rRNA of Pectinatus frisingensis is shown inSEQ ID NO: 2.

EXAMPLE 3 Detection of Pectinatus frisingensis by the PCR method

(1) Selection and synthesis of a primer for Pectinatus frisingensis Thesequences specific for Pectinatus frisingensis by using DNASIS(tradename of Hitachi Soft Engineering Ltd., Co.) on the basis of SEQ IDNO: 1 and SEQ ID NO: 2 were analyzed. The result selected a sequence of377^(th) to 395^(th) on the gene sequence of the spacer region betweenthe gene coding 16S rRNA and the gene coding 23S rRNA of Pectinatusfrisingensis of SEQ ID NO: 1, and a sequence of 195^(th) to 213^(th) onthe gene sequence of the spacer region between the gene coding 16S rRNAand the gene coding 23S rRNA of Pectinatus frisingensis of SEQ ID NO: 2.(SEQ ID NO: 5.)

In addition, the similar analysis selected a sequence of 361^(st) to380^(th) on the gene sequence of the spacer region between the genecoding 16S rRNA and the gene coding 23S rRNA of Pectinatus frisingensisof SEQ ID NO: 1, and a sequence of 179^(th) to 198^(th) on the genesequence of the spacer region between the gene coding 16S rRNA and thegene coding 23S rRNA of Pectinatus frisingensis of SEQ ID NO: 2. (SEQ IDNO: 6.)

Further, specific primer showing in SEQ ID NO: 10 was selected by a genesequence coding 16S rRNA of Pectinatus frisingensis. Theoligonucleotides were chemically synthesized by the same method as inExample 2-()1) .

(2) Detection and identification of Pectinatus frisingensis by theprimers having the sequences of SEQ ID NO: 6 and SEQ ID NO: 10.

The DNA solutions of bacteria prepared in Example 1 were treated withthe primers synthesized in Example 3 (SEQ ID NO:6 and SEQ ID NO:10) byPCR. The temperature conditions of the PCR were as follows:

Thermal denaturation; 94° C., 30 seconds

Annealing; 55° C., 30 seconds

Chain elongation reaction; 72° C., 30 seconds

One cycle of the conditions was repeated 35 times. After the PCR, thereactant was electrophoresed with agalose gel at constant 100 V for 30minutes. A pHY marker was also electrophoresed at the same time as amolecular weight marker. After the electrophoresis, the agarose gel wasstained in about 0.5 μg/ml of an ethidium bromide solution for 20minutes, and ultraviolet was applied to observe the gel and take aphotograph of the gel. By the observation or the photography of the gel,the base length of the amplified products was determined from therelative migration distance of the molecular marker.

As shown in FIG. 1, bands of about 700 bps and about 900 bps weredetected only in case of Pectinatus frisingensis.

From the results, when the oligonucleotides of SEQ ID NO: 6 and SEQ IDNO: 10 were used as PCR primers, the bands having objective length weredetected only in case of Pectinatus frisingensis. Accordingly, it wasshown that each oligonucleotide of the present invention correctlyrecognized the gene sequences of the spacer region between the genecoding 16S rRNA and the gene coding 23S rRNA of Pectinatus frisingensis,and the base sequence targeted on the gene coding 16S rRNA. Moreover,the bands having the aimed length were not observed in the same genusPectinatus cerevisiiphilus, and relative strictly anaerobic bacteria andGram-positive bacteria. Accordingly, Pectinatus frisingensis can bespecifically detected, and at the same time also determined by thepresent invention.

EXAMPLE 4

Cloning and determination of the base sequence of the spacer regionsbetween the gene coding 16S rRNA and the gene coding 23S rRNA ofPectinatus cerevisiiphilus

(1) Selection and synthesis of oligonucleotide primers for amplifying16S123S rRNA spacer regions by PCR

As the base sequence of 16S ribosome RNA gene of Pectinatuscerevisiiphilus is disclosed in International Journal of SystematicBacteriology, Vol. 40, pages 19-27 (1990), the primers were selected onthe basis of the base sequence of 557^(th)-576^(th).

The base sequence of 23 ribosome RNA gene of Pectinatus cerevisiiphilushad not been disclosed, but the base sequence of 23 ribosome RNA gene ofPectinatus frisingensis had been disclosed in Systematic AppliedMicrobiology, Vol. 15, pages 487-501 (1990), EMBL Accession NumberX48423. The primer was selected to obtain the complementary sequencecorresponding to the base sequence of 1^(st)-20^(th) of 23 ribosome RNAgene of Pectinatus frisingensis. Sawaday Technology was entrusted withthe synthesis.

(2) Amplification of 16S/23S rRNA by PCR

The DNA solution 0.1 μg of Pectinatus cerevisiiphilus prepared inExample 1 was charged in a 0.2 ml tube (Perkin-Elmer Co.), 5 μl of 10×buffer in rTaq DNA Polymerase Kit (TOYOBO Co., Ltd.), 3 ,μl of 25 mMMgCl₂, 5 μl of 2 mM dNTP mixture solution (dATP, dGTP, dCTP and dTTP),0.5 μg of 5 unit/μ 1rTaq-polymerase, each 0.5 μl of the 100 mM primersprepared in Example 2-(1) were added, and sterilized water was added toobtain final volume of 50 μl. The tube was set in a thermal cycler of anautomatic gene amplification device (Perkin-Elmer Co.) and amplificationreaction was conducted. 30 cycles were carried under the reactionconditions of one cycle of denaturation at 94° C. for 2.5 minutes,denaturation at 94° C. for 30 seconds, primer annealing at 55° C. for 30seconds and synthetic reaction at 72° C. for 30 seconds. After thereaction, 5 μl of the reactant was used in agarose gel electrophoresis,the DNA was stained with ethidium bromide, and the amplified DNA wasobserved. As a result, DNA of about 1700 bp (abbreviated as “long”) andDNA of about 1400 bp (abbreviated as “short”) were amplified.

(3) Cloning and sequencing of the spacer region “long”

Using a high pure PCR product purification kit (BaringerManhaim),unreactive dNTPs was removed from the solution after the PCR reaction.To 10 ng of the resulting amplified DNA, 2 μl of plasmid pCR 2.1contained in a TA cloning kit (INVITROGEN), 1 μl of ligase and 1 μl ofbuffer were added, and then sterilized water was added to obtain thetotal volume of 10 μl. After the solution was reacted at 14° C. for 4hours, 2 μl of the solution and 2 μl of 0.5 M β-mercaptoethanol wereadded to Escherichia coliINVα′F competent cells, and placed in ice for30 minutes. Then, the solution was heated at 42° C. for 30 seconds, andplasmid transformation to the bacteria was conducted. To the transformedbacteria, 250 μl of a SOC culture (2.0% Tryptone, 0.5% yeast extract,10.0 mM NaCl, 2.5 mM KCl, 10.0 mM MgCl₂—6H₂O, and 20.0 mM glucose) wasadded, and the mixture was shaked at 37° C. for 60 minutes, thentransferred to a LB plate culture medium containing 50 μg/ml ofampicillin and 40 μg/ml X-Gal, and cultured at 37° C. overnight. Theexpressed white colony was transferred to 3 ml of a LB liquid culturemedium containing 50 μg/ml of ampicillin, and cultured at 37° C.overnight.

After the cultivation, plasmids were extracted from the bacteria with aplasmid mini kit (QIAGEN). Apart of the resulting plasmids was taken outand reacted with a restriction enzyme EcoRI (manufactured by TakaraShuzo) at 37 ° C. for 60 minutes, and separated by agaroseelectrophoresis. The DNA was dyed with ethidium bromide, and insertionof “long” was confirmed. 500 ng of the residual plasmid was reacted withrestriction enzyme SmaI (manufactured by TOYOBO Co., Ltd.) at 30° C. for60 minutes. To the reactant, 2 μl of 3 M sodium acetate and 500 μl of100% ethanol were added, and the mixture was placed in ice for 15minutes and centrifuged at 15000 rpm for 15 minutes, and the supernatantwas removed. To the precipitate, 500 μl of 70% ethanol was added, themixture was centrifuged at 15000 rpm for 15 minutes, and thesupernatant, was removed, and the residual was dried for 10 minutesunder reduced pressure. Sterilized water was added to dissolve theprecipitate, and the mixture was reacted with restriction enzyme Xbal(Baringer Manhaim) at 37° C. for 60 minutes. To the reactant, equivalentphenol 11 chloroform (equivalent mixture liquid) was added and gentlymixed, the mixture was centrifuged at 15000 rpm for 15 minutes, and thewater layer (upper layer) was recovered. To the recovery liquid,equivalent water-saturated ether was added and gently mixed, and themixture was centrifuged at 15000 rpm for 15 minutes to remove the etherlayer (upper layer). To the remaining water layer, 2 μl of 3M sodiumacetate and 500 μl of 100% ethanol were added, and the mixture wasplaced in ice for 15 minutes and centrifuged at 15000 rpm for 15 minutesto remove the supernatant.

To the precipitate, 500 μl of 70% ethanol was added, and the mixture wascentrifuged at 15000 rpm for 15 minutes to remove the supernatant, andthe residue was dried under reduced pressure for 10 minutes, and 20 μlof sterilized distillation water was added. To 5 μl of the solution, 1μl of 10× buffer contained in a blunting kit (Takara Shuzo Co., Ltd.)and 3 μl of sterilized distillation water were added, and the mixturewas maintained at 70° C. for 5 minutes, 1 μl of T4 DNA polymerase wasadded, and the mixture was maintained at 37° C. for 5 minutes to obtainblunt ends. After T4 DNA polymerase was inactivated by stirring, 40 μlof ligation solution A and 10 μl of ligation solution B were added, andthe mixture was maintained at 16° C. for 30 minutes to conduct internalligation. 2 μl of the reactant and 2 μl of 0.5M β-mercaptoethanol wereadded to Escherichia coli INVα′F competent cells, and the mixture wasplaced in ice for 30 minutes and heated at 42° C. for 30 seconds, andthe plasmid was transformed to the Escherichia coli.

To the transformed Escherichia coli, 250 μl of a SOC culture medium(2.0% Tryptone, 0.5% Yeast extract, 10.0 mM NaCl, 2.5 mM KCl, 10.0 mMMgCl₂—6H₂O, 20.0 mM glucose) was added, and the mixture was shaken at37° C. for 60 minutes and spread on a LB plate culture medium containing50 μg l/ml of ampicillin to culture at 37° C. overnight. Appeared whitecolonies were inoculated into 3 ml of a LB liquid culture mediumcontaining 50 μg/ml of ampicillin and cultured at 37° C. overnight.After the culture, the plasmid was extracted from the Escherichia coliwith a plasmid mini kit (QIAGEN Company).

Using such obtained plasmid as a template, a sequence reaction wasconducted. As the sequencing primers, an IDRD41 Infrared Dye Labeled M13Forward primer and an IRD41 Infrared Dye Labeled M13 Reverse primer(manufactured by Nisshinbo, sold by Aroka Co., Ltd.) were used. As thereaction liquid, SequiTherm (trademark) Long-Read (trademark) CycleSequencing Kit-LC (manufactured by EPICENTRE TECHNOLOGIES) was used.4000L Long ReadIR (trademark) DNA Sequencing System (manufactured byLI-COR) was used for the determination of the base sequences.

The gene sequence of spacer region “long” between the gene coding 16SrRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus is shownin SEQ ID NO: 3.

(4) Cloning and sequencing of spacer region “short”

Using a high pure PCR product purification kit (Baringer Manhaim),unreactive dNTPs was removed from the solution after the PCR reaction inExample 4-(2). To 100 ng of the resulting amplified DNA, 2 μl of plasmidpCR 2.1 contained in a TAcloning kit (INVITROGEN), 1 μl of ligase and 1μl of buffer were added, and then sterilized water was added to obtainthe total volume of 10 μl. After the solution was reacted at 14° C. for4 hours, 2 μl of the solution and 2 μl of 0.5 M β-mercaptoethanol wereadded to Escherichia coli INVα′F competent cells, and placed in ice for30 minutes. Then, the solution was heated at 42° C. for 30 seconds, andplasmid transformation to the bacteria was conducted. To the transformedbacteria, 250 μl of a SOC culture 2.0% Tryptone, 0.5% yeast extract,10.0 mM NaCl, 2.5 mM KCl, 10.0 mM MgCI₂—6H₂O, and 20.0 mM glucose) wasadded, and the mixture was shaked at 37° C. for 60 minutes, thentransferred to a LB plate culture medium containing 50 μg/ml ofampicillin and 40 μg/ml X-Gal, and cultured at 37° C. overnight. Theappeared white colony was transferred to 3 ml of a LB liquid culturemedium containing 50 μg/ml of ampicillin, and cultured at 37° C.overnight. After the cultivation, plasmid was extracted from thebacteria with a plasmid mini kit (QIAGEN).

A part of the resulting plasmid was taken out and reacted with arestriction enzyme EcoRI (manufactured by Takara Shuzo) at 37° C. for 60minutes, and the reactant was separated by agarose electrophoresis. TheDNA was dyed with ethidium bromide, and insertion of “short” wasconfirmed. 500 ng of the residual plasmid was reacted with restrictionenzyme SmaI (manufactured by TOYOBO Co., Ltd.) at 30° C. for 60 minutes.To the reactant, 2 μl of 3 M sodium acetate and 500 μl of 100% ethanolwere added, and the mixture was placed in ice for 15 minutes andcentrifuged at 15000 rpm for 15 minutes, and the supernatant wasremoved. To the precipitate, 500 μl of 70% ethanol was added, themixture was centrifuged at 15000 rpm for 15 minutes, and the supernatantwas removed, and the residue was dried for 10 minutes under reducedpressure. Sterilized water was added to dissolve the precipitate, andthe mixture was reacted with restriction enzyme BamHI (Takara Shuzo Co.)at 37° C. for 60 minutes. To the reactant, equivalent phenol/chloroform(equivalent mixture liquid) was added and gently mixed, the mixture wascentrifuged at 15000 rpm for 15 minutes, and the water layer (upperlayer) was recovered. To the recovery liquid, equivalent water-saturatedether was added and gently mixed, and the mixture was centrifuged at15000 rpm for 15 minutes to remove the ether layer (upper layer). To theremaining water layer, 2 μl of 3M sodium acetate and 500 μl of 100%ethanol were added, and the mixture was placed in ice for 15 minutes andcentrifuged at 15000 rpm for 15 minutes to remove the supernatant.

To the precipitate, 500 μl of 70% ethanol was added, and the mixture wascentrifuged at 15000 rpm for 15 minutes to remove the supernatant, andthe residue was dried under reduced pressure for 10 minutes, and 20 μlof sterilized distilled water was added. To 5 μl of the solution, 1 μlof 10× buffer contained in a blunting kit (Takara Shuzo Co., Ltd.) and 3μl of sterilized distilled water were added, and the mixture wasmaintained at 70° C. for 5 minutes, 1 μl of T4 DNA polymerase was added,and the mixture was maintained at 37° C. for 5 minutes to obtain bluntends. After T4 DNA polymerase was inactivated by stirring, 40 μl ofligation solution A and 10 μl of ligation solution B were added, and themixture was maintained at 16° C. for 30 minutes to conduct internalligation. 2 μl of the reactant and 2 μl of 0.5M β-mercaptoethanol wereadded to a Escherichia coli INVα′F competent cell, and the mixture wasplaced in ice for 30 minutes and heated at 42° C. for 30 seconds, andthe plasmid was transformed to the Escherichia coli To the transformedEscherichia coli, 250 μl of a SOC culture medium (2.0% Tryptone, 0.5%Yeast extract, 10.0 mM NaCl, 2.5 mM KCl, 10.0 mM MgCl₂—6H₂O, 20.0 mMglucose) was added, and the mixture was shaken at 37° C. for 60 minutesand spread on a LB plate culture medium containing 50 μg/ml ampicillinto culture at 37° C. overnight.

Appeared white colonies were inoculated into 3 ml of a LB liquid culturemedium containing 50 μg/ml of ampicillin and cultured at 37° C.overnight. After the culture, the plasmid was extracted from theEscherichia coli with a plasmid kit (QIAGEN Company).

Using such obtained plasmid as a template, a sequence reaction wasconducted. As the sequencing primers, an IRD41 Infrared Dye Labeled M13Forward primer and an IRD41 Infrared Dye Labeled M13 Reverse primer(manufactured by Nisshinbo, sold by Aroka Co., Ltd.) were used. As thereaction liquid, SequiTherm (trademark) Long-Read (trademark) CycleSequencing Kit-LC (manufactured by EPICENTRE TECHNOLOGIES) was used.4000L Long ReadIR (trademark) DNA Sequencing System (manufactured byLI-COR) was used for the determination of the base sequences.

The gene sequence of spacer region “short” between the gene coding 16SrRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus is shownin SEQ ID NO: 4.

EXAMPLE 5 Detection of Pectinatus cerevisiiphilus by the PCR method

(1) Selection and synthesis of a primer for Pectinatus cerevisiiphilus

The sequences specific for Pectinatus cerevisiiphilus using DNASIS(tradename of Hitachi Soft Engineering Ltd., Co.) on the basis of SEQ IDNO: 3 were analyzed. The result selected a sequence of 135^(th) to153^(rd) on the gene sequence of the spacer region between the genecoding 16S rRNA and the gene coding 23S rRNA of Pectinatuscerevisiiphilus of SEQ ID NO: 3. (SEQ ID NO: 7.)

In addition, the similar analysis selected a sequence of 172^(nd) to191^(st) on the gene sequence of the spacer region between the genecoding 16S rRNA and the gene coding 23S rRNA ofPectinatuscerevisiiphilus of SEQ ID NO: 3. (SEQ ID NO: 8.)

The similar analysis also selected a sequence of 203^(rd l to) 222^(nd)on the gene sequence of the spacer region between the gene coding 16SrRNA and the gene coding 23S rRNA of Pectinatus cerevisiiphilus of SEQID NO: 3. (SEQ ID NO: 9.)

Further, specific primer showing in SEQ ID NO: 11 was selected by a genesequence coding 16S rRNA of Pectinatus cerevisiiphilus. Theoligonucleotides were chemically synthesized by the same method as inExample 2-(1).

(2) Detection and identification of Pectinatus cerevisiiphilus by theprimers having the sequences of SEQ ID NO: 7 and SEQ ID NO: 11.

The DNA solutions of bacteria prepared in Example 1 were treated withthe primers synthesized in Example 5-(1) (SEQ ID NO: 7 and SEQ ID NO:11) by CR. The temperature conditions of the PCR were as follows:

Thermal denaturation; 94° C., 30 seconds

Annealing; 55° C., 30 seconds

Chain elongation reaction; 72° C., 30 seconds

One cycle of the conditions was repeated 35 times. After the PCR, thereactant was electrophoresed with agalose gel at constant 100 V for 30minutes. A pHY marker was also electrophoresed at the same time as amolecular weight marker. After the electrophoresis, the gel was stainedwith 5 μ/ml of an ethidium bromide solution for 20 minutes, andultraviolet was applied to observe the gel and take a photograph of thegel. By the observation or the photography of the gel, the base lengthof the amplified products was determined from the relative migrationdistance with a molecular weight marker.

As shown in FIG. 2, a band of about 600 bps was detected only in case ofPectinatus cerevisiiphilus.

From the results, when the oligonucleotides of SEQ ID NO: 7 and SEQ IDNO: 11 were used as PCR primers, the band having objective length wasdetected only in case of Pectinatus cerevisiiphilus. Accordingly, it wasshown that each oligonucleotide of the present invention correctlyrecognized the gene sequences of the spacer region between the genecoding 16S rRNA and the gene coding 23S rRNA of Pectinatuscerevisiiphilus, and the base sequence targeted on the gene coding 16SrRNA. Moreover, the bands having the aimed length were not observed inthe same genus Pectinatus frisingensis, and relative strictly anaerobicbacteria and Gram-positive bacteria. Accordingly, Pectinatuscerevisiiphilus can be specifically detected, and at the same time alsodetermined by the present invention.

By the present invention, the genes of the spacer region constitutedbetween the 16S rRNA genes and the 23S rRNA genes of Pectinatusfrisingensis and Pectinatus cerevisiiphilus have been proved, and amethod for quickly and reliably detecting Pectinatus frisingensis andPectinatus cerevisiiphilus can be provided by using a part or all of thegene sequences.

11 1 624 DNA Pectinatus frisingensis 1 gaagtcgtaa caaggtagcc gtatcggaaggtgcggctgg atcacctcct ttctaaggat 60 taaaacaatc cgtcgagcac atccggaacatgtattgttt ggttttgagg gtttctccct 120 caaaaaaata gatagaacta atgggggcgtagctcagctg ggagagcacc tgccttgcaa 180 gcagggggtc aggagttcaa atctcctcgtctccaccaga agagaaatgg gcctatagct 240 cagctggtta gagcgcacgc ctgataagcgtgaggtcagt agttcaagtc tacttaggcc 300 caccataatt gcacattgaa aactacacagaagaaaagca aagaacaatt aatcaccaat 360 gccaaacttg tgagaggaga ttttcaagaggatggcgggg aatagttgga ccaagcacaa 420 ttaggaaact aaaaacaagc taagacaaaacatataaact taagctaaag gtgatattct 480 ggaggagact cgagaatata ataaacttaccagaagcgtt cagatgcaag gaagcatgaa 540 agcgaatgaa gaaggcgtat tagtatacgccgatgagtga gctgaaatga tgacgaagca 600 gatgagcggt tatggaaagt ttaa 624 2442 DNA Pectinatus frisingensis 2 gaagtcgtaa caaggtagcc gtatcggaaggtgcggctgg atcacctcct ttctaaggat 60 taaaacaatc cgtcgagcac atccggaacatgtattgttt ggttttgagg gtttctccct 120 caaatattgc acattgaaaa ctacacagaagaaaagcaaa gaacaattaa tcaccaatgc 180 caaacttgtg agaagagatt ttcaagaggatggcggggaa tagttggacc aagcacaatt 240 aggaaactaa aaacaagcta agacaaaacatataaactta agctaaaggt gatattctgg 300 aggagactcg agaatataat aaacttaccagaagcgttca gatgcaagga agcatgaaag 360 cgaatgaaga aggcgtatta gtatacgccgatgagtgagc tgaaatgatg acgaagcaga 420 tgagcggtta tggaaagttt aa 442 3 724DNA Pectinatus cerevisiiphilus 3 gaagtcgtaa caaggtagcc gtatcggaaggtgcggctgg atcacctcct ttctaaggat 60 ttgacaaaaa tctgtcgagt acatccggaatatgtattgt ttggttttga gggtttctcc 120 ctcataaata tatagtagat acttgtaagagtgtttatgg tatgtttaaa agctggtcgg 180 aaatattgtg gtgcaaaaaa atgcatggcagtagagaaga ctggtaaaaa aagaatgaac 240 taatgggggc gtagctcaga tgggagagcacctgccttgc aagcaggggg tcaggagttc 300 aactctcctc gtctccacca gaagagaaagggcctatagc tcagctggtt agagcgcacg 360 cctgataagc gtgaggtcag tagttcaagtctacttaggc ccaccaatat tgcacattga 420 aaactacaca gaagaaagca aagaacaattatcaccaatg ccaaacttgt aagagaaatc 480 gaggagagaa tggcggggaa tagttggaccaagcacaaat taggaaaaga aacaaacgct 540 aagaaacaaa catataaact taagcgaaaaggtgatattc tggaggaaac ttcagagtat 600 ataaacttac cagaagcgtt cagatgcgaggaagggcaaa gctgagagaa gaaagcgtat 660 taatatacgc tgatgaacga agcaaagcactgacaaagca gatggatggt tatgggaagt 720 taca 724 4 399 DNA Pectinatuscerevisiiphilus 4 gaagtcgtaa caaggtagcc gtatcggaag gtgcggctgg atcacctcctttctaaggat 60 ttgacaaaaa tctgtcgagt acatccggaa tatgtattgt ttggttttgagggtttctcc 120 ctcataaata ttgcacattg aaaactacac agaagaaagc aaagaacaattatcaccaat 180 gccaaacttg taagagaaat cgagaagaga atggcgggga atagttggaccaagcacaaa 240 ttaggaaaag aaacaaacgc taagaaacaa acatataaac ttaagcgaaaaggtgatatt 300 ctggaggaaa cttcagagta tataaactta ccagaagcgt tcagatgcgaggaagggcaa 360 agcactgaca aagtagatgg atggttatgg gaagttaca 399 5 19 DNAPectinatus frisingensis 5 ccatcctctt gaaaatctc 19 6 20 DNA Pectinatusfrisingensis 6 tctcrtctca caagtttggc 20 7 19 DNA Pectinatuscerevisiiphilus 7 cactcttaca agtatctac 19 8 20 DNA Pectinatuscerevisiiphilus 8 ccacaatatt tccgaccagc 20 9 20 DNA Pectinatuscerevisiiphilus 9 agtcttctct actgccatgc 20 10 20 DNA Pectinatusfrisingensis 10 cgtatccaga gatggatatt 20 11 20 DNA Pectinatuscerevisiiphilus 11 cgtatgcaga gatgcatatt 20

What is claimed is:
 1. An isolated nucleic acid comprising SEQ ID NO:3.
 2. An isolated nucleic acid comprising SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.
 3. A method for detecting Pectinatus cerevisiphilus comprising carrying out a gene amplification assay wherein a nucleic acid comprising instant SEQ ID NO: 3 is produced as an amplification product, and detecting the presence of the amplification product, wherein the presence of instant SEQ ID NO: 3 is indicative of the presence of Pectinatus cerevisiphilus.
 4. A method for detecting Pectinatus cerevisiphilus comprising using an isolated nucleic acid according to claim 2 as a primer for synthesis of nucleic acids, and treating the nucleic acid by gene amplification to detect the bacteria. 